| According to the global cancer statistical analysis data,the incidence of esophageal cancer ranks seventh in malignant tumors and the overall mortality rate ranks sixth.In China,although the treatment regimen of surgery combined with chemotherapy has been matured and applied to the clinic,the effect is not ideal,and the incidence and mortality of esophageal cancer are still increasing year by year.Therefore,it is important to find more effective therapeutic targets for esophageal cancer.In the development of malignant tumors,angiogenesis plays an important role in the rapid growth and metastasis of tumors.Therefore,targeted angiogenesis is an effective way to treat tumors.At present,some anti-angiogenesis targeted drugs have been applied to clinical practice as treatment strategies for various tumors and improve the survival outcome of clinical patients.However,there have been relatively few clinical trials of anti-angiogenic therapies in esophageal cancer.Therefore,there is an urgent need for further research and development of new therapies against angiogenesis in esophageal cancer.YAP acts as a key downstream effector of the Hippo pathway,exerting the role of oncogenes.Recent studies have shown that YAP plays an important role in tumor angiogenesis.Therefore,targeting YAP may be considered a new strategy for treating esophageal cancer.Verteporfin is a YAP inhibitor that inhibits tumor cell proliferation,migration and angiogenesis by disrupting YAP-TEAD interactions,and induces apoptosis.In addition,VP can also inhibit tumor angiogenesis in retinoblastoma and pancreatic ductal adenocarcinoma.However,the role of VP in regulating esophageal cancer progression and tumor angiogenesis is unclear.This project will investigate the role of the YAP inhibitor Verteporfin in the malignant progression of esophageal cancer and drug resistance,and explore whether it can be achieved by targeting the tumor angiogenesis microenvironment.Objective:1.To explore the effect of YAP inhibitor Verteporfin on the malignant progression of esophageal cancer cells.2.To explore the effect of Verteporfin on tumor angiogenesis.3.To investigate Verteporfin reverses esophageal cancer paclitaxel resistance by targeting tumor angiogenesis.Methods:1.Cytotoxicity assay,colony formation assay,DAPI staining,Western blot,wound healing assay and invasion assay were used to observe the effects of different concentrations of Verteporfin(0,0.6,1.2,2.4 μM)on the malignant progression of esophageal cancer cells KYSE150 and KYSE30.2.Cytotoxicity assay,colony formation assay,DAPI staining,wound healing assay and angiogenesis assay in vitro and in vivo were used to investigate the effects of different concentrations of Verteporfin(0,0.375,0.75,1.5 μM)on proliferation,apoptosis,migration and angiogenesis of HUVEC.Angiogenesis assay in vitro and in vivo,tumor-endothelial cell adhesion and chemotaxis assay and Western blot detection of angiogenesis-related molecules were used to investigate the effect of different concentrations of Verteporfin(0,0.6,1.2,2.4 μM)on angiogenesis mediated by esophageal cancer cells KYSE150 and KYSE30.3.Low-dose induction method to construct low-dose and moderate-dose drug resistant esophageal cancer cells.Through cytotoxicity assay,colony formation assays,angiogenesis assay in vitro and in vivo were used to observe the effects of 0.05 μM paclitaxel and 0.6 μM Verteporfin alone and in combination on the proliferation and angiogenesis of esophageal cancer cells with different degrees of resistance to paclitaxel.Results:1.After different concentrations of Verteporfin were treated with KYSE150 and KYSE30,the cytotoxicity assays and colony formation assays showed that compared with the control group,the proliferation rate of the cells after the drug treatment was significantly decreased,and the number of colony formed was also reduced(P < 0.05).DAPI staining showed that Verteporfin led to nuclear condensation,and even appear apoptotic body.Western blot displayed that the protein expression level of pro-caspase-3 was significantly reduced(P < 0.05).Wound healing and invasion assays showed that Verteporfin weakened the ability of cell migration and invasion(P< 0.05).2.After HUVECs were treated with different concentrations of Verteporfin,the cytotoxicity assay and the colony formation assay showed that the cell proliferation rate and number of colony formed decreased significantly(P < 0.05).DAPI staining showed that Verteporfin resulted in nuclear condensation and Western blot displayed that the protein expression level of pro-caspase-3 was significantly reduced(P < 0.05).Wound healing assays showed that Verteporfin concentration is inversely correlated with cell migration ability(P < 0.05).Angiogenesis assay in vitro and in vivo showed that Verteporfin can inhibit the angiogenesis ability of HUEVC and esophageal cancer.Tumor-endothelial cell adhesion and chemotaxis assay showed that Verteporfin inhibited the angiogenesis of esophageal cancer by reducing the adhesion and chemotaxis of tumor cells and endothelial cells(P < 0.05).Western blot results showed that Verteporfin may inhibit esophageal cancer angiogenesis through NF-κB/β-catenin/VEGFA/MMP-2 signaling pathway.3.Through cytotoxicity assays,it was confirmed that drug-resistant cell lines of esophageal cancer were successfully constructed.The inhibitory effect of the combination of paclitaxel and Verteporfin on drug-resistant esophageal cancer cells was more obvious than that of the drug alone.Through the colony formation assay,it was found that the number of colony decreased when 0.05 μM paclitaxel and 0.6 μM Verteporfin alone were used to treat esophageal cancer drug-resistant cells,and the combined effect was more significant(P < 0.05).Angiogenesis assay in vitro and in vivo showed that the combination of paclitaxel and Verteporfin inhibited tumor angiogenesis in drug-resistant cells.Conclusions:1.The YAP inhibitor Verteporfin can inhibit the malignant progression of esophageal cancer cells and promote cell apoptosis.2.Verteporfin can inhibit the proliferation,migration,in vitro and in vivo angiogenesis ability of HUVEC,and promote cell apoptosis.At the same time,it can also inhibit the in vitro and in vivo angiogenesis ability of esophageal cancer cells by attenuating the adhesion and chemotaxis between esophageal cancer cells and HUVEC.3.Verteporfin combined with paclitaxel can reverse esophageal cancer drug resistance by inhibiting tumor angiogenesis. |
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